Stimulation of an immune response is dependent upon the presence of antigens recognized as foreign by the host immune system. Bacterial antigens such as Salmonella enterica and Mycobacterium bovis BCG remain in the phagosome and stimulate CD4 T-cells via antigen presentation through major histocompatibility class II molecules. In contrast, bacterial antigens such as Listeria monocytogenes exit the phagosome into the cytoplasm. The phagolysosomal escape of L. monocytogenes is a unique mechanism which facilitates major histocompatibility class I antigen presentation of listerial antigens. This escape is dependent upon the pore-forming sulfhydryl-activated cytolysin, listeriolysin O (LLO).
The ability of L. monocytogenes to break down the vacuole within a host cell and enter the cytoplasm has led to its use as a recombinant vaccine. U.S. Pat. No. 5,830,702 describes vaccines comprising attenuated mutants of Listeria spp. genetically engineered to express foreign antigens in the cytoplasm of infected macrophages and other cells. Several approaches for expressing the antigen in Listeria spp. are described including generation of a fusion protein of a selected foreign antigen and a listerial protein, preferably an enzyme involved in lysis of host vacuoles. In particular, a fusion protein encoding the hly promoter and the first 416 amino acids of LLO fused in-frame to the entire coding sequence of the NP antigen was constructed in E. coli and on transformation to Listeria monocytogenes is demonstrated to secrete a 105 kDA protein that reacts with antiserum to LLO and NP (col. 24 of '702 patent). Recombinant L. monocytogenes secreting a fusion protein comprising listeriolysin O and NP (LLO-NP) was demonstrated to target infected cells for lysis by NP-specific class I-restricted cytotoxic T cells. In contrast, a hemolysin-negative L. monocytogenes strain expressing LLO-NP presented the antigen in a class II restricted manner (Ikonimidis et al. J. Exp. Med. 1994 180:2209-2218). Thus, from these studies it was surmized that hemolysin-dependent bacterial escape from the vacuole is necessary for class I presentation in vitro.
The escape function of L. monocytogenes has also been transferred to Bacillus subtilis and attenuated Salmonella ssp. strains (Bielecki, J. et al. Nature 1990 345:175-176, Gentschev et al. Infect. Immun. 1995 63:4202-4205). S. enteric and M. bovis BCG vaccine carriers which secrete listeriolysin O have also been constructed (Kaufmann, S. H. and Hess, J. Immunol. Lett. January 1999 65(1-2):81-4). These constructs are taught to be capable of introducing antigens into the MHC class II and MHC class I pathway, resulting in stimulation of both CD4 and CD8 T-cells. Comparison of S. enterica vaccines which display the same listerial antigen in secreted and somatic form showed the secreted antigen display to be superior to the somatic antigen display (Kaufman, S. H. and Hess, J. Immunol. Lett. January 1999 65(1-2):81-4).
WO 99/10496 discloses recombinant BCG strains secreting hemolytically active hly with an improved MHC class I-restricted immune response for use as a vaccine against tuberculosis.
Administration of purified listeriolysin O encapsulated in liposomes has also been reported to be effective in the induction of antigen-specific Th1-dependent protective immunity to various kinds of intracellular parasitic bacteria in vivo (Tanabe et al. Infect. Immun. February 1999 67(2):568-75).
It has now been found that the immune response to an antigen can be enhanced by fusion of the antigen to a non-hemolytic truncated form of listeriolysin O (ΔLLO).